DamID Profiling of CTCF and TEAD3 Binding within Chromosome 9p21
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Date
2018-05
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The Ohio State University
Abstract
The progression of benign tumor growth is often restricted by oncogene-induced senescence (OIS), a state of irreversible cell cycle arrest that frequently involves upregulation of the p16INK4a tumor suppressor. Senescence is ultimately controlled by the accumulation of p16INK4a, but the intermediary steps which cause p16INK4a transcriptional upregulation are unknown. Here, I employ the DNA adenine methyltransferase identification (DamID) assay to investigate protein binding to DNA elements within the INK/ARF locus, which encodes p16INK4a. In DamID, an E. coli Dam methylase is fused to DNA-binding factors of interest. Interaction of the Dam-fused DNA binding factor with DNA causes local adenosine methylation. Patterns of DNA binding can then be investigated using a methylation-sensitive restriction enzyme digest followed by qPCR for the region of interest. I selected putative INK/ARF binding proteins and made three fusion constructs: Dam-CTCF, Dam-TEAD3, and Dam-SMAD3. I mapped the steady-state binding patterns for each of these fusion proteins across the 90kb INK/ARF regulatory region. In this way, I quantified the usage of several known binding sites and identified novel interactions. With this knowledge, we can now investigate how oncogene activation remodels DNA-binding factor interactions to engage, and eventually subvert, the tumor suppressive functions of the INK/ARF locus.
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Keywords
INK/ARF, DamID, 9p21, Cancer, OIS, Senescence